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Challenges Direct, non-invasive measurement of the properties of live, functioning neurons remains a technical challenge. Optical methods, while otherwise ideal, have faced two major hurdles

a) many of these native biomolecules respond only in the ultraviolet (UV), and

b) aggregation states of the molecules are not resolvable within the theoretical limits of optical resolution.

Multiphoton microscopy (MPM) can non-destructively harness UV fluorescence (Maiti et al. 1997a), and fluorescence correlation spectroscopy (FCS) can measure particle sizes down to sub-nanometer levels (Maiti et al., 1997b). We have now refined these techniques so that they are able to tackle these hurdles.

Serotonin Imaging:

We are able to capture three-photon images of neurons with adequate resolution to show the serotonin vesicles. We are using this capability to study the dynamics of serotonin release induced by depolarisation in immortalised raphe neurons. We are also studying the release dynamics associated with the action of amphetamines in immortalised neurons, neuorns in primary culture and in rat brain slices. Another area of effort involves studying the changes in the vesicular content and distribution as a function of differentiation.

Protein aggregation:

We have employed FCS to measure diffusional transport (and therefore the hydrodynamic size) of protein aggregates and vesicles. We have investigated the aggregation of b -amyloid (implicated in Alzheimer's disease).

We have used FCS to investigate receptor oligomerization on membranes of chinese hamster ovary cells, and have developed a new method for analyzing FCS data in such complex systems.

Spectroscopy:

We have determined the multiphoton absorbance and photsostability properties of tryptophan and other chromophores, using techniques developed in-house.

We have achieved an order of magnitude improvement in the sensitivity for detecting tryptophan (serotonin precursor) by utilizing novel excitation sources.

Instrumentation:

We have built a UV enhanced multi-photon microscopy for imaging the autofluorescence of monoamines in live neurons. We have also built a combined multi-photon/single-photon fluorescence correlation spectrometer for studying protein aggregation. We are in the process of combining a patch-clamp electrophysiology set-up with a multi-photon microscope.

We have demonstrated an insertable confocal probe, which has the potential to perform many of these studies in vivo.

We have developed an alginment less fluorescence correlation microscope, that helps in performing FCS on living cells.

We have extended the spectral range of the multiphoton microscope to detect deep UV fluorophores (~ 300 nm) by incorporating a non-epifluorescent detector. We have determined the two photon excitation spectra of catecholamines dopamine and nor-epinephrine using the non-epifluorescent detector. Currently we are exploring the possibility of imaging dopamine in live neurons

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P.hd in BiophotonicsTata Institute of Fundamental Research

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